As the nucleic acid sequences of a number of genomes, including the human genome, become available, there is an increasing need to interpret this wealth of information. While the availability of nucleic acid sequence information allows for the prediction and identification of genes, it does not explain the expression patterns of the proteins produced from these genes. The genome does not describe the dynamic processes on the protein level. For example, the identity of genes and the level of gene expression does not represent the amount of active protein in a cell nor does the gene sequence describe post-translational modifications that are essential for the function and activity of proteins. Thus, in parallel with the genome projects there has begun an attempt to understand the proteome (i.e., the quantitative protein expression pattern of a genome under defined conditions) of various cells, tissues, and species. Proteome research seeks to identify targets for drug discovery and development and provide information for diagnostics (e.g., tumor markers).
An important aspect of genome and proteome analysis is the ability to differentiate expression patterns between two related samples (e.g., differentiated and undifferentiated cells, cancer cells and normal cells, drug-treated cells and untreated cells, etc.). The importance of such techniques can be seen by looking at the example of cancer cells. An important current area of research involves developing an understanding of the mechanisms behind cancer progression. In order to follow changes in cancer cells at the molecular level, methods are used that monitor the activation of different genes as the cancer process evolves. This is usually performed by monitoring mRNA expression using techniques such as differential display (Liang and Pardee, Science 257:967 [1992] and Miller et al., Electrophoresis 20:256 [1999]) and subtractive hybridization (Schweinfest and Papas, Intern. J. Oncol., 1:499 [1992]). The differential display method is based upon the systematic amplification of portions of mRNAs, which are then resolved on a DNA sequencing gel. On the other hand, the subtractive hybridization method works by subtracting cDNAs reverse transcribed from mRNA from two physiological states. This allows for the isolation of transcripts that are differentially expressed. The isolated transcripts then undergo a series of hybridization reactions followed by selective amplification. Even though these methods provide information on gene activation, there are inherent problems with them (Sturtevant, Clin. Micro. Rev., 13:408 [2000]). Since the methodology depends upon amplification of rare transcripts by PCR, results are semi-quantitative at best, where the ability to study quantitative changes is often important. Also, bands that are differentially displayed in one trial are often difficult to reproduce in a second run and differential expression is often difficult to confirm by Northern blotting. However, often the mRNA is altered without a corresponding change observed in protein levels, and protein levels are frequently altered without a corresponding change observed in mRNA levels (Russel et al., Oncogene 18:1983 [1999] and Ozturk et al., Anal. Cell Pathol. 16:201 [1998]).
The problems involved with correlating changes in cancer cells to mRNA expression have led investigators to study altered protein expression in cancer progression. Since proteins are the basic entities that perform functions in the cells, it becomes logical to follow changes in protein expression as cells progress to malignancy. This involves using methods to monitor changes in quantitative expression of proteins and also structural changes in proteins during progression. The classic methods for following such changes in protein expression involve 1-D and 2-D polyacrylamide-gel electrophoresis. The 1-D gel method is generally a simple method used to achieve a crude separation of cell lysates where the most abundant proteins can be separated and detected. Although a relatively low resolution technique, 1-D gel method remains a general method for monitoring the more highly expressed proteins in cells. 2-D gel electrophoresis is a high resolution method capable of separating out hundreds of protein spots, where the spot pattern is characteristic of the cell protein expression. 2-D gel patterns have been traditionally used to study changes in proteins that are peculiar to stages of cancer progression (Lopez, Electrophoresis 21:1082 [2000]; Langen, Electrophoresis 21:2105 [2000]; and Williams et al., Electrophoresis 19:333 [1998]).
Gel electrophoresis methods (1-D and 2-D) have certain fundamental limitations for screening and identification of proteins from cells. Gel electrophoresis separations are slow, where even a 1-D gel requires nearly eight hours to run with bands having sufficient resolution to study protein changes. Also, gel electrophoresis only provides separation, where for proteins that change in expression, identification of the proteins is required. Although various procedures have been developed for identifying proteins based upon MALDI-MS of in-gel digests (Shevchenko et al., Anal. Chem., 68:850 [1996]; Courchesne et al., Electrophoresis 18:369 [1997]; Aebersold et al., Proc. Natl. Acad. Sci. USA 84:6970 [1987]; Waltham et al., Electrophoresis 18:391 [1997]; and Henzel et al., Proc. Natl. Acad. Sci., USA 90:5011 [1993]), the procedures remain rather labor intensive and laborious. In addition, direct determination of the molecular weight of intact proteins from gels remains difficult, although there have been several new developments for molecular weight determination (Loo et al., Anal. Chem., 68:1910 [1996]; Cohen and Chait, Anal. Biochem., 247:257 [1997] and Liang et al., Anal. Chem., 68:1012 [1996]). Another significant problem with gel electrophoresis is quantitation, where small changes in expression (plus or minus 10%) are often difficult to observe with Coomassie staining, and quantitation at any level is difficult with silver staining (Rodriguez et al, Electrophoresis 14:628 [1993]). Other methods are required to routinely screen for changes in protein expression and identification. Thus, what is needed are new methods and systems to allow efficient and informative comparison of protein expression patterns between cells (e.g., cancer and normal cells).